Project Summary p53 is a transcription factor that plays a crucial role in tumor suppression, and it is frequently mutated in human cancers. Missense mutations in the DNA binding domain of p53 can result in a gain-of-function phenotype, leading to increased cell proliferation and tumor formation. While mutations in other tumor suppressor proteins such as RB1 and NF1 are deletion mutations leading to decreased protein expression, point mutations in p53 lead to increased p53 stability and prolonged half-life. In order to improve and develop novel treatments for cancers containing mutant p53 (mtp53), its mechanisms for increased stability and accumulation in tumors needs to be largely expanded. Specifically, whether mtp53 requires critical interacting proteins or posttranslational modifications for its increased accumulation and gain-of-function effect remains to be elucidated. Most work in identifying novel interactors or modifications on mtp53 has relied on targeting specific signaling pathways that modulate p53 activity. However, mtp53 is regulated by a wide array of external signals, so a comprehensive and unbiased approach in studying its regulation is crucial in understanding its mechanisms. Therefore, this proposal aims to utilize a quantitative mass spectrometry-based approach in identifying interactors and modifications across different mtp53 mutations and cancer types. This proposed research will reveal a new layer of information that will aid in determining mechanisms of mtp53 stability, and it will lead to the discovery of novel targets for cancer therapeutics. This proposal elucidates the mechanisms of mtp53 through two Aims: (1) Identifying and validating mtp53 interactors across multiple cancer cell lines harboring GOF mtp53 using immunoprecipitation coupled to mass spectrometry, and determining whether the candidate ?stabilizers? are essential for cancer growth using CRISPR gRNA screening approaches. (2) Identifying combinatorial posttranslational modifications essential for mtp53 stability by developing a quantitative mass spectrometry method for mtp53. Overall, these aims will shed light on the mechanisms of mtp53 stability in human cancers, which will offer promising therapeutic targets in cancer.